Alkaline phosphatase as a label for immunoassay using amperometric detection with a variety of substrates and an optimal buffer system

Novel substrates for use in an amperometric 3-electrode system are describe d for the determination of alkaline phosphatase (EC.3.1.3.1), the enzyme la bel most commonly used in electrochemical immunoassays. Previous problems e ncountered with a variety of substrates have led to passivation of the work ing electrode at low product concentrations. Our group has synthesised a nu mber of novel substrates in an attempt to elucidate this fouling problem. T hese substrates were synthesised with various electron withdrawing groups a t the para position as these groups seem to stabilise the reaction product in an alkaline medium [I. Rosen, J. Rishpon, J. Electroanal. Chem. 258 (198 9) 27-39] and Limit fouling of the working electrode [Y. Xu, H.B. Halsall, W.R. Heineman, J. Pharm. Biomed. Anal. 7 (12) (1989) 1301-1311]. These subs trates were p-cyanophenylphosphate, p-methoxyphenylphosphate and p-formylph enylphosphate. Preventing the formation of phenolic radicals, which polymer ise and adhere to the working electrode, is the primary goal when designing the synthesis of these substrates. Of these, p-cyanophenylphosphate showed the greatest stability of product, significant lowering of the fouling of the working electrode and comparable Michaelis-Menten kinetics. Four aminoe thanol buffers were compared with respect to their abilities to stabilise t he products and to promote enzyme activity. 0.1 M Tris(hydroxymethyl)aminom ethane buffer, at pH 9.0, was found to be the optimum buffer system. (C) 19 99 Elsevier Science B.V. All rights reserved.